Creutzfeldt-Jakob disease (CJD) is the human form of a group of neurodegenerative diseases collectively known as transmissible spongiform encephalopathies (TSE). These conditions are characterized by high levels of the abnormal form prion protein (PrP) i.e., PrP-res, and have counterparts in bovine spongiform encephalopathy (BSE) and scrapie in sheep. The infectious agents for these conditions have not been identified, but it is known that the infection can be transmitted through the food supply as well as through peripheral routes including white blood cells in experimental animals and humans. Other animals besides humans, sheep and cattle have also been affected by the same or analogous agents, including pigs, rodents, primates, cats and various zoo animals. See, for example, Manuelidis, L., Science (1997) 277:94-98. There is no known treatment for these conditions and because humans are at risk with respect to this TSE, the human form is particularly of interest. It would be helpful to provide a high throughput assay to evaluate protocols and compositions that would be useful in treating or preventing this condition in humans and other mammals. The present invention employs successful cell lines as models for the various, including human, forms of this disease.
Cell lines have been prepared from neuroblastoma cell lines that are infected with a scrapie agent which appear to be unstable. Race, R., Curr. Top. Microbiol. Immunol. (1991) 172:181-193; Borchelt, D., et al., J. Cell Bio. (1990) 743-752. Further, the characteristics of these cells as indicative of infection appear to be unreliable, although these cultures are useful in elucidating metabolism of PrP-res and removal of PrP-res after treatment with selected compounds. See, Priola, S., et al., Infect. Agents Dis. (1994) 23:54-58; Vey, M., et al., Proc. Natl. Acad. Sci. USA (1996) 93:14945-14949; and Mange, A., et al., J. Virol. (2000) 74:3135-3140. Only infectivity related to scrapie has been evaluated in these cell lines, however. The murine hypothalamic GT1 and N2a cell lines have proved more stable with respect to scrapie, but only with specific strains. Bosque, P., et al., J. Virol. (2000) 74:4377-4386.
The present invention resides in creating cell lines stably expressing the TSE phenotype, including the appearance of PrP-res. These cell lines are useful in assays to identify compounds that will be effective in treating or preventing the development of TSE, e.g., CJD in humans. Previous reports of reproducibly infected murine hypothalamic GT1-7 cells (hereafter called GT cells) with a number of different scrapie and CJD agents that had been serially passaged in mice are set forth in Nishida, N., et al., J. Virol. (2000) 74:320-325; Arjona, A., et al., Proc. Natl. Acad. Sci. USA (2004) 101:8768-8773. Stable infection of GT cells did not require cell cloning.
As will be evident below, the invention, in addition to providing cell lines stably infected with a TSE agent for evaluating candidate treatments or for preparing large amounts of the agent itself, also concerns assays to identify potential vaccine candidates as well as methods for identifying a particular strain of agent based on previously observed behavior. These assays reflect the ability of weaker forms of an infectious agent to effect a sort of immunity with regard to more virulent forms, analogous to, for example, vaccination against smallpox, or the use of attenuated virus as a vaccine against polio. Previous work in vivo indicates that similar effects may be seen in TSE infective agents. For example, two strains of CJD have been identified—an attenuated SY strain and a more virulent FU strain. Manuelidis, L., et al., Proc. Natl. Acad. Sci. USA (2003) 100:5360-5365. The ability of the SY strain to protect against infection by the FU strain was demonstrated in mice (ibid.).
In TSEs such as human Creutzfeldt-Jakob Disease (CJD), sheep scrapie, and bovine spongiform encephalopathy (BSE), typical cellular and adaptive immune responses to a foreign infectious agent have not been detected (Manuelidis, L., et al., Science (1997) 277:94-98). Thus the discovery that SY was able to prevent superinfection by the more virulent and rapidly lethal FU agent in mice was surprising (Manuelidis, L., Proc. Natl. Acad. Sci. USA (1998) 95:2520-2525). FU and SY are CJD agents isolated from geographically separate regions of the world. When passaged in mice, these agents were readily distinguished by profound differences in the incubation time to disease, as well as by the distribution of brain lesions. The attenuated “slow” SY agent, propagated from a sporadic CJD case, produced only small medial thalamic lesions, whereas the virulent “fast” FU strain caused widespread severe lesions.
Even with an intracerebral route of inoculation, there was an obvious protective effect of SY against high doses of FU challenge agent. Control mice, first inoculated with uninfected brain and then with FU, all developed widespread FU disease >110 days before any clinical signs were seen in SY protected mice (Manuelidis (1998), supra), and representative brains of SY protected mice failed to show even trace FU agent (Manuelidis, L., et al., Neurosci. Lett. (2000) 293:163-166). Moreover, when lower doses of SY doses preceded FU challenge, mice lived without any TSE disease for their entire normal lifespan of >600 days. In contrast, parallel mock inoculated mice all died prematurely of fulminant FU disease 325 days earlier. Similarly clear protective effects of SY were also seen with peripheral routes of infection (Manuelidis (2003), supra).
No comparable interference has been verified using other TSE strains (Manuelidis (1998), supra). Only a small −35 day increase in incubation time was observed with the scrapie strains 22L the 22A inoculated intracerebrally in mice (Dickinson, A., et al., Nature New Biol. (1972) 237:244-245). This raised the possibility that the SY CJD agent possessed a unique ability to protect the host.
It would be helpful to have a convenient assay system to determine the possibility that weaker strains of TSE agents can exert a protective effect on infection by more virulent ones. The stably infected cell lines of the invention provide such a high throughput system.